Use of therapeutically useful peptides

The present invention relates to preventing and treating endothelial dysfunction by using biologically active peptides and products containing them. A product having a high short-chain peptide content has been found especially effective for use in accordance with the present invention.

The product to be used in accordance with the present invention can be formulated for instance as a health and wellness food product or a pharmaceutical product. It contains small-molecular peptides, such as the tripeptides Ile-Pro-Pro (IPP), Val-Pro-Pro (VPP), or mixtures or concentrates containing them, and it functions by improving epithelial function and curing diseases relating thereto. A specific aspect of the present invention is to reduce stiffness and thus enhance elasticity of blood vessels with the use of small-molecular peptides, such as tripeptides Ile-Pro-Pro (IPP), Val-Pro-Pro (VPP), or mixtures, concentrates and other products containing the same.

The vascular endothelium regulates locally vascular tone by the release of vasodilator substances, such as endothelium-derived relaxing factor (EDRF), nitric oxide, prostacyclin and endothelium-derived hyperpolarizing factor (EDHF) and vasoconstrictor substances, such as thromboxane A₂, free radicals and endothelin. The importance of nitric oxide in both basal and stimulated control of vascular tone in large epicardial coronary arteries and in the coronary microcirculation has been shown in several clinical studies (for review see Vapaatalo H, Mervaala E. Clinically important factors influencing endothelial function. Med Sci Monit 2001; 7(5):1075-1085. Drexler H, Hornig B. Endothelial dysfunction: a novel therapeutic target. J Mol Cell Cardiol 1999; 31:51-60). Above-mentioned different endothelial derived relaxing factors correct endothelial dysfunction e.g. in atherosclerosis. Regulation of vasomotor tone, modulation of blood coagulation, promotion and prevention of vascular growth, modulation of inflammation and action as a target to the effects and adverse effects of drugs are associated to endothelium. EDRF release is stimulated by increased flow in vessels of for example bradykinin, thrombin, acetylcholine and serotonin.

The endothelium controls underlying smooth muscle tone in response to certain pharmacological and physiological stimuli. The endothelial function plays also a role in vascular growth, leukocyte adhesion, and immunological regulation, metabolism of circulating amines, lipoprotein metabolism and integration and transduction of blood-borne signals.

Endothelial dysfunction is characterized by decreased secretion of vasodilatory mediators, increased production of vasoconstrictors, increased sensitivity to vasoconstrictors and/or resistance of vascular smooth muscle to endothelial vasodilators. Dysfunction is a consequence of an imbalance between relaxing and contracting factors, or growth promoting and inhibiting agents. Inflammation, lipoprotein oxidation or other oxidative stress reactions are factors affecting development and maintenance of endothelial dysfunction. Theoretically, the clearest and most direct indicators of endothelial dysfunction are nitric oxide and its metabolites, as well as cyclic GMP.

Endothelial dysfunction can be either a cause or a consequence of several clinical conditions, such as hypertension, atherosclerosis, coronary disease, heart failure, diabetes and high blood cholesterol. The most direct indicators of endothelial dysfunction are imbalance between decreased production or receptor function of vasodilatory factors, such as nitric oxide, prostacyclin, endothelium derived hyperpolarizing factor (EDHF), and natriuretic peptides, or increased formation of or sensitivity to vasoconstrictive agents, such as endothelin-1, angiotensin 11, endoperoxides, and thromboxane A₂.

Endothelial dysfunction can be treated with several known drugs, the most important being angiotensin converting enzyme (ACE) inhibitors, angiotensin II receptor blockers, nitrate preparations and cholesterol lowering drugs. The effect of ACE inhibitors is mainly based on their capability to improve the effect of bradykinin, which enhances the synthesis of nitric oxide in endothelium. Induced and/or enhanced nitric oxide production or added nitrates can balance insufficient internal nitric oxide production. The medicines and nitrates function as exogenic EDRF and dilate blood vessels, and in addition they are active as antithrombotic compound in damaged veins.

WO 02/34767 A1, Selwood et al., describes peptides which are fragments of vascular endothelial growth factor (VEGF), and useful for inhibiting angiogenesis. According to the invention, the peptides comprise three to eight amino acids, and a key feature of the amino acid sequence is the presence and arrangement of basic residues, in particular arginine (Arg) and/or lysine (Lys) residues. The document specifies several peptides, consisting of from six to sixteen amino acids. None of them include the sequences Isoleucin-Proline-Proline (IPP) or Valine-Proline-Proline (VPP). According to the publication, the peptides may be useful in diseases where angiogenesis plays a significant role in pathology. Such diseases may include diabetic retinopathy, age-related macular degeneration (ARDS), cancer, endometriosis, psoriasis and rheumatoid arthritis.

WO 99/45941, Sandberg et al., describes a composition used to enhance the softness, elasticity, or appearance of tissue. The composition is formulated from peptides that correspond to any one of 41 peptide fragments produced from thermolysing digestion of elastin. Preferably, the composition comprises a polypeptide having the formula R1-Valyl-Valyl-Prolyl-Glutamine-R2, wherein R1 is an amino portion of the peptide, and R2 is a carboxy portion of the peptide. The composition is preferably applied to human skin in a cosmetic formulation. According to the document, the composition may also be useful for treating hypertension, coronary heart disease, arteriosclerosis, angina, coronary thrombosis, chronic obstructive pulmonary disease, and restenosis post angioplasty.

WO 01/91700 A2 and U.S. Pat. No. 6,962,904 B1, both Mitts et al, are in part based on the same study as WO 99/45941, Sandberg et al. The documents describe compositions for enhancing the elasticity of tissue, and the compositions are formulated from peptides corresponding to sequences found in elastin, in particular the sequences -Valine-Valine-Proline- and -Valine-Valine-Proline-Asparagine-. Said compositions are useful for improving elastin production in tissues. According to the publications, the main utility is once again in cosmetics, but it is also mentioned that the compositions may be useful in treating e.g. hypertension, coronary heart disease, arteriosclerosis, angina, coronary thrombosis, chronic obstructive pulmonary disease and restenosis post-angioplasty.

The described elastin peptide fragments contain a large number of glycine and/or proline residues as compared to other amino acid sequences. The fragments do not include the sequences Isoleucin-Proline-Proline (IPP) and/or Valine-Proline-Proline (VPP).

Preparation of bioactive peptides by fermentation has been largely studied and described in the background art, one particular point of interest being milk-derived peptides. These have been shown to have for instance opioid receptor binding properties, ACE inhibiting activity and antimicrobial properties. Several studies have been made in relation to e.g. hypertension, but effects of the peptides on the endothelial functions have not been described. In particular, effects of the tripeptides Valine-Proline-Proline (VPP) and Isoleusine-Proline-Proline (IPP) and products containing the same on the endothelial functions have not been described in the background art.

The tripeptides VPP and IPP are known compounds, which have been described as ACE inhibitors having an antihypertensive effect. For instance in J Dairy Sci 78 (1995) 777-783, Nakamura et al. describe the use of a starter containing Lactobacillus helveticus and Saccharomyces cerevisiae for the preparation of two ACE inhibitors. The compounds were both identified as tripeptides, Val-Pro-Pro and Ile-Pro-Pro. Although the publication does not describe an in vivo antihypertensive effect of the tripeptides, it is mentioned to be the next subject of research.

U.S. Pat. No. 5,449,661, Nakamura et al., discloses the preparation of a peptide containing the tripeptide sequence Val-Pro-Pro and its use for lowering high blood pressure. The peptide is prepared by fermenting fat-free milk powder with the Lactobacillus helveticus strain JCM-1004, whereafter the peptide is purified chromatographically and freeze-dried.

In WO 99/16862, Yamamoto et al. describe the Lactobacillus helveticus strain CM4, FERM BP-6060, which is capable of producing a large amount of the tripeptides Val-Pro-Pro and/or Ile-Pro-Pro.

WO 01/32905, Valio, describes a product having antihypertensive properties and its preparation. The product is produced by fermenting a casein-containing starting material with a lactic acid bacterium, and performing nanofiltration on the obtained, peptide-containing fermentation product. The antihypertensive properties of the product are in part due to the tripeptides IPP and VPP contained therein. WO 03/070267, Valio, describes the use of IPP and VPP, as well as the product described in WO 01/32905, in the preparation of a product enhancing the availability of minerals. The product can be used e.g. for increasing bone formation, strengthening the skeleton system and for treating or prevention of osteoporosis.

Blood vessels have implications in diseases associated with viscoelasticity, including hypertension, arteriosclerosis, angina, angiogenesis, myocardial infarction, coronary thrombosis, restenosis post angioplasty, and chronic obstructive pulmonary disease. Cardiovascular diseases are amongst the most common diseases in the world, and they are on the top five list of life threatening diseases in many countries. Unfortunately, increasing living standards also increase the risk of said diseases, and hence they will play an even greater role in the future. In addition to conventional drugs, functional products are nowadays providing an attractive alternative to the consumers.

Functional products improving the elasticity of blood vessels and improving and normalizing endothelial function would hence be very welcome as part of a regular diet. Furthermore, administering peptides with beneficial arterial stiffness properties as a medical or pharmaceutical product is also worth considering.

It is thus an objective of the present invention to make available a product that as part of a regular diet, or as a medical and pharmaceutical product, improves or normalizes endothelial function and hence is capable of preventing, alleviating or curing disorders and diseases relating to endothelial dysfunction. Endothelial dysfunction has a remarkable role in the stiffness or flexibility of blood vessels, which in turn is important in many severe disorders including e.g. coronary heart disease, arteriosclerosis, angina, coronary thrombosis, chronic obstructive pulmonary disease and restenosis post-angioplasty. Hence, ability to enhance the elasticity of blood vessels is an especially important feature of the present invention.

The product to be used according to the invention consists of or comprises peptides improving endothelial function.